Key Laboratory of Clinical Medicine Henan Universities, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450003, China.
Key Laboratory of Immunology and Kidney Disease, People's Hospital of Henan Province, Zhengzhou University, Zhengzhou, 450003, China.
Int J Med Sci. 2020 Jun 5;17(10):1406-1414. doi: 10.7150/ijms.45603. eCollection 2020.
Adoptive cellular immunotherapy employing chimeric antigen receptors-modified T (CAR-T) cells has demonstrated promising antitumor effects in hematologic cancers. However, CAR-T therapy confront many challenges in solid tumors like immunosuppressive microenvironment, molecular heterogeneity, etc. The cancer genome atlas (TCGA) of hepatocellular carcinoma (HCC) revealed many genetic characteristic and molecular tumorigenesis. EGFRvIII is a tumor specific antigen widely expressed in a variety of cancers including HCC and an ideal therapeutic target for cancer therapy. The liver cancer cell line SMMC7721 express high level EGFRvIII and widely applied in HCC investigations. Herein, we developed EGFRvIII CAR-T cells by piggyBac transposon system, and detected its specific killing effect against SMMC7721 cells and . Results indicated that transduction efficiency of CAR reached 53.1%. Expression of CAR protein was verified by immunoblotting as a band of approximate 57KD. The killing effect of CAR-T cells against SMMC7721 was positively correlated with E/T ratio (E:T=5:1, 10:1, 20:1, 40:1), and exceeded 50% at 20:1 ratio. Significant increase in IFN-γ and TNF-α secretion were detected in the co-culture supernatant of CAR-T cells and SMMC7721, comparable to the level of exogenous EGFRvIII-expressing U87 cells. The killing activity and cytokine secretion were both dependent on the expression level of EGFRvIII in target cells. In HCC xenograft models, CAR-T cells could effectively suppress the growth of SMMC7721. In conclusion, EGFRvIII CAR-T cells demonstrated specific antitumor effect against SMMC7721 and , providing basis for immunotherapy of HCC in future clinical use.
嵌合抗原受体修饰 T 细胞（CAR-T）的过继细胞免疫疗法在血液系统恶性肿瘤中显示出有希望的抗肿瘤作用。然而，CAR-T 疗法在实体瘤中面临许多挑战，如免疫抑制微环境、分子异质性等。肝癌（HCC）的癌症基因组图谱（TCGA）揭示了许多遗传特征和分子肿瘤发生。表皮生长因子受体变体 III（EGFRvIII）是一种广泛表达于多种癌症（包括 HCC）的肿瘤特异性抗原，是癌症治疗的理想治疗靶点。肝癌细胞系 SMMC7721 表达高水平的 EGFRvIII，广泛应用于 HCC 研究。在此，我们通过 piggyBac 转座子系统构建了 EGFRvIII CAR-T 细胞，并检测了其对 SMMC7721 细胞和 的特异性杀伤作用。结果表明，CAR 的转导效率达到 53.1%。免疫印迹法验证了 CAR 蛋白的表达，其条带约为 57KD。CAR-T 细胞对 SMMC7721 的杀伤作用与 E/T 比值呈正相关（E:T=5:1、10:1、20:1、40:1），在 20:1 比值时超过 50%。在 CAR-T 细胞与 SMMC7721 的共培养上清中检测到 IFN-γ和 TNF-α的分泌显著增加，与外源性表达 EGFRvIII 的 U87 细胞相当。杀伤活性和细胞因子分泌均依赖于靶细胞中 EGFRvIII 的表达水平。在 HCC 异种移植模型中，CAR-T 细胞能有效抑制 SMMC7721 的生长。综上所述，EGFRvIII CAR-T 细胞对 SMMC7721 和 具有特异性抗肿瘤作用，为未来临床 HCC 免疫治疗提供了依据。
